Difference between revisions of "Part:BBa K2505010"
| Line 26: | Line 26: | ||
==Material and Method== | ==Material and Method== | ||
===Reagent assay=== | ===Reagent assay=== | ||
| − | 1. - Cultivate Receiver E.coli in LB medium containing antibiotics for about 15hours | + | 1. - Cultivate Receiver <i>E.coli</i> in LB medium containing antibiotics for about 15hours |
2. - Dilute the culture to 1/200 with flesh LB medium containing antibiotics | 2. - Dilute the culture to 1/200 with flesh LB medium containing antibiotics | ||
| Line 39: | Line 39: | ||
===Supernatant Assay=== | ===Supernatant Assay=== | ||
| − | 1. - Cultivate Sender <i>E.coli<> in LB medium for about 15hours in 37℃ or 25℃ | + | 1. - Cultivate Sender <i>E.coli</i> in LB medium for about 15hours in 37℃ or 25℃ |
2. - Centrifuge the culture 16,000rpm and 5minutes | 2. - Centrifuge the culture 16,000rpm and 5minutes | ||
Revision as of 14:04, 28 October 2017
Ptet-rbs-traI-tt
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 465
- 1000COMPATIBLE WITH RFC[1000]
This part constitutively produces C8.
Characterization
Material and Method
Reagent assay
1. - Cultivate Receiver E.coli in LB medium containing antibiotics for about 15hours
2. - Dilute the culture to 1/200 with flesh LB medium containing antibiotics
3. - Incubate the flesh culture for 2 hours
4. - Mix 495μL of the culture with 5μL of DMSO solution (each DMSO is containing 100 microM,10microM...of AHL to reach final concentration 1microM 100nM...) in micro tube
5. - Incubate the micro tube for 5 hours with Small shaking incubator in 37℃
6. - Take 100μL of culture and Measure fluorescent (excitation wave length is 495nm, Measurement wavelength is 520nm) and absorbance (Measurement wavelength is 600nm)
Supernatant Assay
1. - Cultivate Sender E.coli in LB medium for about 15hours in 37℃ or 25℃
2. - Centrifuge the culture 16,000rpm and 5minutes
3. - Follow Reagent assay process (1~4) and Prepare Reporter culture.
4. - Mix 250µL of sender culture’s supernatant with Reporter culture in micro tube.
5. - Incubate the micro tube for 5 hours with Small shaking incubator in 37℃
6. - Take 100μL of culture and Measure fluorescent (excitation wave length is 495nm, Measurement wavelength is 520nm gain is 45) and absorbance (Measurement wavelength is 600nm)
Result
The protein(LuxR) is Receptor for C6 signals. But it is known LuxR bind to other AHL such as C10 and cause crosstalk. We confirmed LuxR respond to C8 signals and have almost same sensitibvity in the case of C6 signals. Receiver E.coli’s RFU (Reletive Fluoroscent Units) in each concentration (1µM,100nM,10nM…) is shown in Figure . Error bar have a same width as standard deviation (n=3). Detection limit is over 10nM in case of C6 and C8. RFU values are almost same over 100nM.